(i) Field of the Invention
The invention relates to plant genetic engineering. In particular, the invention relates to methods and compositions for modulating properties of fruit dehiscence in plants, particularly Brassicaceae plants, specifically to improved methods and means for reducing seed shattering, or reducing seed shattering until after harvest, in plants such as Brassicaceae plants, particularly the Brassicaceae plants grown for oil production, to a degree which is agronomically important.
(ii) Description of the Related Art
Siliques or pods from Brassicaceae plants release their seeds through a process called dehiscence. A silique consists of two carpels joined margin to margin. The suture between the margins forms a thick rib, called replum. As pod maturity approaches, the two valves separate progressively from the replum, along designated lines of weakness in the pod, eventually resulting in the shattering of the seeds that were attached to the replum. The dehiscence zone defines the exact location of the valve dissociation.
Shedding of seed (also referred to as “seed shatter” or “pod shatter”) by mature pods, before or during crop harvest, is a universal phenomenon with crops that develop dry dehiscent fruits. Premature seed shatter results in a reduced seed recovery, which represents a problem in crops which are grown primarily for the seeds, such as oil producing Brassicaceae plants, particularly oilseed rape. Another problem related to premature seed shattering is an increase in volunteer growth in the subsequent crop year. In oilseed rape, pod-shatter related yield losses typically range from 10 to 25%, but can reach up to 50%, depending on the weather conditions.
Current commercial oilseed rape varieties are extremely susceptible to shattering. Kadkol et al. [(1986), Aust. J. Biol. 34: 79] reported increased resistance towards shattering in a single, Australian accession of rape. Variation in pod maturation has further been observed in mutants of rape stemming from irradiated seeds [Luczkiewicz (1987), Proc. 7th Int. Rapeseed Congress 2: 463]. However the traditional methods for breeding have been unsuccessful in introducing shatter resistance into rape cultivars, without interference in other desirable traits such as early flowering, maturity and blackleg resistance [Prakash and Chopra (1990), Genetical Research 56: 1].
Several genes, which promote or inhibit pod dehiscence have been identified in Arabidopsis thaliana through mutant analysis. These genes are encoding putative MADS box and basic helix-Loop-Helix mutants. Combined mutants in both SHATTERPROOF1 (SHP1; initially referred to as AGL1) and SHATTERPROOF2 (SHP2; initially referred to as AGL5) result in indehiscent siliques (i.e. siliques which remain closed upon maturity in Arabidopsis thaliana) (Liljegren et al., 2000, Nature 404, 766-770). Similarly, mutants in the INDEHISCENT gene in Arabidopsis thaliana (PCT publication WO 01/79517), as well as in ALCATRAZ (Rajani et al. 2001, Current Biology 11, 1914-1922) interfered with pod dehiscence leading to podshatter resistance. Constitutive expression of FRUITFUL (FUL), a repressor of SHP and IND, in Arabidopsis thaliana also resulted in indehiscent siliques (Ferrandiz et al., 2000, Science, 289, 436-438). These transcription factors are thus regulating the development of the valves, including the valve margins and the dehiscence zone.
Genes for a number of hydrolytic enzymes, such as endopolygalacturonases, which play a role, during pod dehiscence, in the programmed breakdown of the dehiscence zone in pods from Brassicaceae plants have also been identified (see e.g. WO 97/13865; Petersen et al., Plant. Mol. Biol., 1996, 31:517-527).
To isolate mutant alleles corresponding to ind, alc or shp1-shp2 in economically important Brassicaceae plants, such as oilseed rape, is a laborious and time consuming task. Moreover, such isolation may be complicated by the amphidiploidy in oilseed rape and the consequent functional redundancy of the corresponding genes.
It has been described that the expression of the ALC, IND, AGL1 and AGL5 genes or their homologues, may be downregulated using gene silencing techniques such as antisense suppression or cosuppression (WO99/00503; WO01/79517; WO0159122).
Vancanneyt et al., 2002 (XIII International Conference on Arabidopsis Research, Sevilla, Spain June 28-Jul. 2; 2002) reported that the expression of FUL from A. thaliana under control of a CaMV 35S promoter in oilseed rape, resulted in a number of podshatter resistant transformants. Pods of such podshatter resistant lines had no dehiscence zone, and opening of the pods could only be achieved by random fracture of the valves by applying considerable pressure.
Vancanneyt et al., 2002 (XIII International Conference on Arabidopsis Research, Sevilla, Spain June 28-Jul. 2; 2002) also reported that silencing of the IND gene in Arabidopsis thaliana using so-called dsRNA silencing techniques resulted in almost complete podshatter resistance. 98% of the transgenic Arabidopsis lines developed siliques, which did not open along the valve suture, and could only be opened by applying considerable pressure to the valves.
Furthermore, experiments described in this application (see below) indicate that silencing of the IND gene in Brassica napus using dsRNA gene silencing techniques, whereby the dsRNA sequence is identical to one of the IND homologues of Brassica napus, results in podshatter resistant lines with pods that bearly open by random fracture of the valves in a standardized random impact test.
It is important to realize that while seed shattering constitutes an important problem in oilseed rape culture, which may be solved by developing podshatter resistant lines, ultimately, separation of the seeds from the pods is still required. In normal agricultural practice this is achieved by threshing of the pods by a combine harvester. Threshing of the pods by a combine harvester must be complete and must cause minimum damage to the seeds thus released. However, as pod strength increases, the more severe action required to thresh them causes an unacceptable level of damage to the seed. The pods of podshatter resistant Brassicaceae plants should thus not be so strong that they cannot be threshed in a combine harvester (Bruce et al. 2001, J. Agric. Engng Res. 80, 343-350).
The art thus remains defective in providing method and means for reducing seed shattering in Brassicaceae plants, particularly in oilseed rape, while retaining a sufficient threshability.
These and other objects are achieved by the present invention, as indicated by the various embodiments described in the summary of the invention, detailed description and claims.